The invention of this application relates to a linker that is able to adjust the translation frame of DNA chain encoding protein. More particularly, it relates to a frame-adjusting linker that is indispensable for various modification of DNA fragments.
Protein is synthesized (translated) in vivo from a messenger RNA (mRNA) transcribed from genomic DNA. For analyzing function of a protein in the area of molecular biology and life science, it is general to express a mutant protein from a mutant DNA fragment prepared by transferring information of mRNA to DNA to give a double-stranded DNA and operating the base sequence of this DNA fragment (cDNA).
In the preparation of deletion or insertion mutant DNA fragment, it is usually carried out that two sites of DNA are cleaved by restriction enzymes to remove the sequence between them or that one site is cleaved and some sequence or base is inserted thereinto.
DNA strand encodes one amino acid residue by a block (codon) of three bases. Therefore, when DNA is cleaved by a restriction enzyme so that a part of sequence or base is deleted or when some sequence or base is inserted to the cleaved site, there is a risk that frame of the codon changes and, as a result, the amino acid sequence of the protein in the C-terminal side from the mutated site is changed to a completely different sequence. Or it is also probable that a stop codon is formed therein and the synthesis of protein is interrupted. In this case, the desired mutant protein cannot be prepared. Therefore, when a mutation such as deletion or addition is introduced into DNA strand, it is necessary to make the translation frame in sequence before and behind the mutation being same.
In general, a linker DNA consisting of double-stranded oligonucleotide is linked at the cleaved site for adjusting the translation frames. However, it is necessary to prepare various kinds of linkers for each time depending upon the type of the restriction enzyme used and, in addition, selection of the linker is not always easy. Therefore, a lot of labor is needed for the preparation of the mutant DNA fragment having correct translation frame.
The invention of this application has been carried out in view of the circumstances of the prior art as mentioned above. The object of the invention is to provide a frame-adjusting linker, which has the sites recognized by plural restriction enzymes, for the preparation of mutant DNA sequences having the correct translation frames before and behind the any cleavage sites.
As a solution for the above-mentioned object, this application provides the following inventions (1) to (4).
(1) A translation frame-adjusting linker, which is a single-stranded DNA consisting of a palindrome base sequence of SEQ ID NO. 1.
(2) The translation frame-adjusting linker claim 1, of which the 5xe2x80x2-terminal is phosphorylated.
(3) A translation frame-adjusting linker, which is a single-stranded DNA consisting of a palindrome base sequence of SEQ ID NO. 2.
(4) The translation frame-adjusting linker of claim 3, of which the 5xe2x80x2-terminal is phosphorylated.